The impact of loss of function DNA sequence variants in the human protocadherin gene cluster on neural circuit assembly.

经修订的项目摘要/摘要： 在发育过程中，人类大脑中约800亿个神经元之间准确地组装了复杂的神经电路。哺乳动物聚集型原钙粘附素(Pcdh)基因编码一系列高度不同的细胞表面同源结合蛋白，为单个神经元提供了正常神经回路组装所需的独特细胞表面身份或条形码。先前对聚集性Pcdh基因和蛋白质的结构、功能和基因表达的研究揭示了Pcdh条形码的产生和功能的复杂机制，以及功能丧失的Pcdh突变对小鼠神经回路组装和行为的影响。Pcdh条形码的一个显著特征是，它由相互作用的神经元细胞膜上的细胞表面蛋白“晶格”组成。Pcdh条形码已被证明在小鼠的轴突自我回避、神经元瓦片和正常行为中发挥关键作用。在细胞培养研究中，已经证实了破坏细胞间相互作用的显性功能丧失的Pcdh突变。 尽管未能准确组装神经回路与神经发育和神经精神障碍有关，但在自闭症谱系障碍(ASD)队列的大规模DNA测序研究中，PCDH基因簇中单个PCDH基因中单核苷酸变体(SNV)的出现并不符合全基因组遗传关联的统计关联标准。然而，在理解PCDH基因簇的调节和功能方面的进展，以及最近在Simons Simplex队列中获得的ASD先证者的淋巴母细胞系(LCL)携带PCDH基因的干扰新生SNV，为进一步探索PCDH基因在人类诱导的多能干细胞来源的神经元中的功能提供了独特的机会。 其目的是对ASD病例中发现的PCDH基因簇进行从头开始SNV的系统分析。在目标1中，我们将在基于细胞的体外亲和结合分析中，确定PCDH亚型中的SNV如何影响PCDH蛋白的同亲相互作用和细胞内定位。在目标2中，我们将产生CRISPR-Cas9编辑的带有PCDH突变缺失的HiPSC，并将它们分化为神经元，以评估HiPSC来源的神经元中自我回避的缺陷。在目标3中，我们将以5-羟色胺能神经元为模型，研究PCDH亚型SNV对小鼠神经回路和功能连通性的影响。这些拟议的研究将极大地促进我们对PCDH基因座在大脑发育过程中神经回路组装中的功能作用的理解。

REVISED PROJECT SUMMARY/ABSTRACT: Complex neural circuits are accurately assembled between the approximately 80 billion neurons in the human brain during development. The mammalian clustered protocadherin (Pcdh) genes, which encode a family of highly diverse cell-surface homophilic binding proteins, provide individual neurons with a unique cell-surface identity, or barcode required for normal neural circuit assembly. Previous structural, functional, and gene expression studies of the clustered Pcdh genes and proteins have revealed complex mechanisms by which Pcdh barcodes are generated and function, as well as the impact of loss of function Pcdh mutations on neural circuit assembly and behavior in mice. A remarkable feature of the Pcdh barcode is that it consists of a cell surface protein “lattice” on the membranes of interacting neurons. The Pcdh barcode has been shown to play a critical role in neurite self-avoidance, neuronal tiling and normal behavior in mice. Dominant loss of function Pcdh mutations that disrupt cell-cell interactions have been demonstrated in cell culture studies. Although the failure to accurately assemble neural circuits has been linked to neurodevelopmental and neuropsychiatric disorders, the occurrence of single nucleotide variants (SNVs) in individual PCDH genes across the PCDH gene cluster do not meet a genome wide statistical association criteria for genetic association in large scale DNA sequencing studies of autism spectrum disorder (ASD) cohorts. However, advances in understanding the regulation and function of the PCDH gene cluster and the recent availability of lymphoblastoid cell lines (LCLs) from ASD probands in the Simons Simplex cohort bearing disruptive de novo SNVs in PCDH genes presents a unique opportunity to further explore PCDH gene function in human neurons derived from human induced pluripotent stem cells. The aims are to conduct a systematic analysis of de novo SNVs across the PCDH gene clusters identified in ASD cases. In Aim 1 we will determine how SNVs in PCDH isoforms impact homophilic interactions and intracellular localization of PCDH proteins in cell-based in-vitro homophilic binding assays. In Aim 2, we will generate CRISPR-Cas9 edited hiPSCs bearing loss of function PCDH mutations and differentiate them into neurons to assess deficits in self-avoidance in the hiPSC-derived neurons. In Aim 3, we will investigate the impact of SNVs in PCDH isoforms on neural circuits and functional connectivity in mice, using serotonergic neurons as a model. These proposed studies should significantly advance our understanding of the functional role of the PCDH locus in neural circuit assembly during brain development.